scholarly journals 13C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis ofLactococcus lactisreveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3451 ◽  
Author(s):  
Kamalrul Azlan Azizan ◽  
Habtom W. Ressom ◽  
Eduardo R. Mendoza ◽  
Syarul Nataqain Baharum
Keyword(s):  
Amino Acids ◽  
Malic Enzyme ◽  
Metabolic Flux ◽  
Pearson Correlation ◽  
Starter Culture ◽  
Flux Ratio ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Ratio Analysis ◽  
Central Metabolism

Lactococcus lactissubsp.cremorisMG1363 is an important starter culture for dairy fermentation. During industrial fermentations,L. lactisis constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response ofL. lactisto several stresses has been described, the adaptation mechanisms at the level ofin vivofluxes have seldom been described. To gain insights into cellular metabolism,13C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism ofL. lactiswhen subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis (r) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability ofL. lactis’central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) inL. lactis, in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering inL. lactis. Overall, the integration of systematic analysis of amino acids and flux ratio analysis provides a systems-level understanding of howL. lactisregulates central metabolism under various conditions.

scholarly journals 13C metabolic flux analysis on roles of malate transporter in lipid accumulation of Mucor circinelloides

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Lu Wang ◽  
Huaiyuan Zhang ◽  
Yao Zhang ◽  
Yuanda Song
Keyword(s):  
Fatty Acid ◽  
Metabolic Flux Analysis ◽  
Lipid Accumulation ◽  
Malic Enzyme ◽  
Metabolic Flux ◽  
Flux Ratio ◽  
Tca Cycle ◽  
Flux Analysis ◽  
Control Strain ◽  
Malate Transporter

Abstract Background Mitochondrial and cytoplasmic malate transporter proteins are responsible for transmembrane transport of malate, thereby linking malate metabolism in various subcellular regions of the cell. These transporters play an important role in fatty acid biosynthesis of oleaginous microorganisms. Our previous studies have found that lipid content of the recombinant Mucor circinelloides (M. circinelloides) strain with mitochondrial malate transporter (mt) gene overexpression was increased by 70%, while that of strain with mt gene knockout was decreased by 27%. However, the mechanism of malate transporter promoting the transport of mitochondrial malate and citrate related to lipid accumulation is not clear. Therefore, 13C-labeled glucose metabolic flux analysis was carried out to identify the metabolic network topology and estimate intracellular fluxes of genetically engineered M. circinelloides strains for the purpose of better understanding the roles of malate transporters in citrate transport systems and lipid accumulation. Results The metabolic flux distribution analysis suggested that tricarboxylic acid (TCA) cycle flux ratio of mt-overexpression strains was decreased compared to that of the control strain, but in contrast, glyoxylic acid (GOX) cycle flux ratio was increased. Accordingly, the mt-knockout strain showed an opposite phenomenon with a higher TCA cycle flux ratio and a lower GOX cycle flux ratio than the control strain. GOX cycle might be more effective than TCA cycle in producing malate and oxaloacetate replenishment. Moreover, a relatively higher flux ratio of the pentose phosphate (PP) pathway was obtained in mt-overexpression strains, but no significant difference in the malic enzyme flux between recombinant strains and the control strain. Our results confirmed that PP pathway might play an important role for supplying NADPH and malic enzyme is not a limiting factor for fatty acid synthesis in oleaginous fungus M. circinelloides strains. Conclusion Intracellular metabolic flux information suggested that mt-overexpression strains had higher flux in PP pathway and GOX cycle, lower flux in TCA cycle, and no difference in malic enzyme cycle. Together, the role of malate transporter was assumed to further participate in transporting cycle of acetyl-CoA and drive PP pathway to supply NADPH required for lipid accumulation in recombinant M. circinelloides strains.

scholarly journals Responses of theCentral Metabolism in Escherichia coli to PhosphoglucoseIsomerase and Glucose-6-Phosphate DehydrogenaseKnockouts

2003 ◽  
Vol 185 (24) ◽  
pp. 7053-7067 ◽  
Author(s):  
Qiang Hua ◽  
Chen Yang ◽  
Tomoya Baba ◽  
Hirotada Mori ◽  
Kazuyuki Shimizu
Keyword(s):  
Escherichia Coli ◽  
Pentose Phosphate Pathway ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Flux Ratio ◽  
Pentose Phosphate ◽  
Phosphoglucose Isomerase ◽  
Overflow Metabolism ◽  
Flux Analysis ◽  
Glucose Catabolism

ABSTRACT The responses of Escherichia coli central carbon metabolism to knockout mutations in phosphoglucose isomerase and glucose-6-phosphate (G6P) dehydrogenase genes were investigated by using glucose- and ammonia-limited chemostats. The metabolic network structures and intracellular carbon fluxes in the wild type and in the knockout mutants were characterized by using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-13C]glucose labeling and two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, glycerol, and glucose. Disruption of phosphoglucose isomerase resulted in use of the pentose phosphate pathway as the primary route of glucose catabolism, while flux rerouting via the Embden-Meyerhof-Parnas pathway and the nonoxidative branch of the pentose phosphate pathway compensated for the G6P dehydrogenase deficiency. Furthermore, additional, unexpected flux responses to the knockout mutations were observed. Most prominently, the glyoxylate shunt was found to be active in phosphoglucose isomerase-deficient E. coli. The Entner-Doudoroff pathway also contributed to a minor fraction of the glucose catabolism in this mutant strain. Moreover, although knockout of G6P dehydrogenase had no significant influence on the central metabolism under glucose-limited conditions, this mutation resulted in extensive overflow metabolism and extremely low tricarboxylic acid cycle fluxes under ammonia limitation conditions.

scholarly journals Fine tuning the glycolytic flux ratio of EP-bifido pathway for mevalonate production by enhancing glucose-6-phosphate dehydrogenase (Zwf) and CRISPRi suppressing 6-phosphofructose kinase (PfkA) in Escherichia coli

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Ying Li ◽  
He Xian ◽  
Ya Xu ◽  
Yuan Zhu ◽  
Zhijie Sun ◽  
...  
Keyword(s):  
Pentose Phosphate Pathway ◽  
Conversion Rate ◽  
Metabolic Flux ◽  
Flux Ratio ◽  
Reducing Power ◽  
Pentose Phosphate ◽  
Fine Tuning ◽  
High Yield ◽  
Glycolytic Flux ◽  
Acetyl Coa

Abstract Background Natural glycolysis encounters the decarboxylation of glucose partial oxidation product pyruvate into acetyl-CoA, where one-third of the carbon is lost at CO2. We previously constructed a carbon saving pathway, EP-bifido pathway by combining Embden-Meyerhof-Parnas Pathway, Pentose Phosphate Pathway and “bifid shunt”, to generate high yield acetyl-CoA from glucose. However, the carbon conversion rate and reducing power of this pathway was not optimal, the flux ratio of EMP pathway and pentose phosphate pathway (PPP) needs to be precisely and dynamically adjusted to improve the production of mevalonate (MVA). Result Here, we finely tuned the glycolytic flux ratio in two ways. First, we enhanced PPP flux for NADPH supply by replacing the promoter of zwf on the genome with a set of different strength promoters. Compared with the previous EP-bifido strains, the zwf-modified strains showed obvious differences in NADPH, NADH, and ATP synthesis levels. Among them, strain BP10BF accumulated 11.2 g/L of MVA after 72 h of fermentation and the molar conversion rate from glucose reached 62.2%. Second, pfkA was finely down-regulated by the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system. The MVA yield of the regulated strain BiB1F was 8.53 g/L, and the conversion rate from glucose reached 68.7%. Conclusion This is the highest MVA conversion rate reported in shaken flask fermentation. The CRISPRi and promoter fine-tuning provided an effective strategy for metabolic flux redistribution in many metabolic pathways and promotes the chemicals production.

Bioreaction Network Topology and Metabolic Flux Ratio Analysis by Biosynthetic Fractional 13C Labeling and Two-Dimensional NMR Spectroscopy

1999 ◽  
Vol 1 (3) ◽  
pp. 189-197 ◽  
Author(s):  
Thomas Szyperski ◽  
Ralf W Glaser ◽  
Michel Hochuli ◽  
Jocelyne Fiaux ◽  
Uwe Sauer ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Network Topology ◽  
Metabolic Flux ◽  
Flux Ratio ◽  
Ratio Analysis ◽  
Two Dimensional ◽  
13C Labeling

scholarly journals Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

2004 ◽  
Vol 70 (12) ◽  
pp. 7277-7287 ◽  
Author(s):  
Christoph Wittmann ◽  
Patrick Kiefer ◽  
Oskar Zelder
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Metabolic Flux ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Phosphotransferase System ◽  
Lysine Production ◽  
Metabolic Fluxes ◽  
Fructose 1,6 Bisphosphate

ABSTRACT Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-13CFru]sucrose, [1-13CGlc]sucrose, and [13C6 Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTSMan or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.

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